Regulation of matrix metalloproteinases in a model of colonic wound healing in a rabbit.

PURPOSE: Matrix metalloproteinases occur in the colon at an anastomosis but not in the normal colon. Matrix metalloproteinase synthesis can be regulated by cytokines, for example interleukin-1 beta and growth factors, such as transforming growth factor beta and basic fibroblast growth factor. The aim of this study was to investigate the regulation of matrix metalloproteinases at an anastomosis by identifying the cell types that synthesize matrix metalloproteinases, examining factors that might regulate their synthesis, determining whether they occur in an active form, and assessing the effect of suture type on these parameters. METHODS: An anastomosis was formed in the distal colon of rabbits using either polyglactin or polydioxanone and the animals were killed six hours or seven days later. The distribution of matrix metalloproteinases and cytokines and the cell types were assessed by immunohistochemistry. Matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were detected also by zymography. RESULTS: Immunohistochemistry showed that matrix metalloproteinases were restricted to the suture line. Although zymography demonstrated that matrix metalloproteinase-2 was present mainly in an active form, matrix metalloproteinase-9 and matrix metalloproteinase-3 were present in the pro-form. The active form of matrix metalloproteinase-3 occurred more often in the polydioxanone-sutured rabbits. With the exception of matrix metalloproteinase-9, the matrix metalloproteinases were synthesized by fibroblasts. Interleukin-1 beta and transforming growth factor beta were more widespread than in the normal colon and were localized adjacent to the matrix metalloproteinases. Basic fibroblast growth factor was also more widespread postoperatively but occurred deeper in the anastomosis than the matrix metalloproteinases. CONCLUSIONS: This study has shown that interleukin-1 beta and transforming growth factor beta may regulate the synthesis of the matrix metalloproteinases by fibroblasts and that minor differences that occur in the matrix metalloproteinase profile are dependent on the suture type.

Effect of colonic obstruction on the distribution of matrix metalloproteinases during anastomotic healing.

BACKGROUND: Anastomotic dehiscence is common after surgery for colonic obstruction. The strength of an anastomosis is dependent on collagen, which is degraded by matrix metalloproteinases (MMPs). The aim of this study was to determine the distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP) 1 in an experimental model of colonic obstruction, with and without resection and anastomosis. METHODS: The distal colon of rabbits was obstructed with a Silastic ring for 24 h and then either the ring was removed or the obstructed segment was resected and an anastomosis formed. Rabbits were killed immediately or at intervals for up to 7 days after operation. The distribution of the MMPs and TIMP-1 was examined by indirect immunofluorescence. RESULTS: MMPs and TIMP-1 were present throughout the descending colon for 24 h in both groups. They persisted to the third day in rabbits with an anastomosis but by day 7 were restricted to the suture line. Their presence correlated with microscopic damage. CONCLUSION: The extensive distribution of the MMPs suggests that these enzymes contribute to anastomotic dehiscence, but only in the immediate postoperative period.

Role of matrix metalloproteinases in healing of colonic anastomosis.

PURPOSE: The aim of this study was to compare the distribution of the matrix metalloproteinases (MMPs) during anastomotic healing in a normal colon with that in an ischemic colon in a rabbit. This family of enzymes degrades all components of connective tissue and has been implicated as a cause of anastomotic dehiscence. METHODS: A left-sided anastomosis was formed in the distal colon of one group of rabbits, and in the other group, 9 cm of distal colon was made ischemic before resection and anastomosis 12 hours later. Tissues from the anastomosis and sites around the colon were removed at 12 hours, 1 day, and 3 days after anastomosis and, also, at 7 days in the normal group. Distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP), was localized by indirect immunofluorescence. RESULTS: In rabbits having only an anastomosis, the MMPs and TIMP-1 were, at all times, seen solely in the anastomotic segment and were strictly confined to the immediate vicinity of the suture line. While in rabbits with an ischemic colon before anastomosis, the MMPs initially extended several centimeters proximally and distally from the suture line. By the third day, however, there were only minor differences between the two models. CONCLUSION: Distribution of the MMPs and TIMP-1 in normal healing is consistent with a role in the remodeling of colonic anastomosis, but when healing of the colon is compromised, these enzymes are more widespread and may contribute to anastomotic dehiscence.

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumor cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Loss of cell-cell and cell-matrix adhesion molecules in colorectal cancer.

Adhesion molecules are thought to play a vital role in the induction and maintenance of tissue differentiation and their loss or down-regulation has been implicated in the neoplastic process. Recent studies have shown that the morphoregulatory activities are a consequence of interactive processes between several cell adhesion molecules rather than the function of a single molecule. Therefore, we have investigated a panel of adhesion molecules including members of the integrin, cadherin and immunoglobin superfamily in colorectal cancer. Twenty-eight consecutive colorectal adenocarcinomas were stained using an avidin-biotin indirect immunoperoxidase technique. Our results showed a consistent loss of the alpha 2 and beta 1 integrin subunits (21/28 = 75% and 22/28 = 78.6% respectively) and a decrease in expression of E-cadherin in 5/5 poorly differentiated adenocarcinomas. Carcinoembryonic antigen expression was preserved but with basolateral accentuation seen in tumours. There was no statistical correlation with Dukes' stage. These results provide further evidence that in colorectal cancer there is a widespread deregulated expression of cell-cell and cell-matrix adhesion molecules. Changes in the expression and function of adhesion molecules which regulate growth and differentiation may play a role in the behaviour of colorectal cancer.

Direct measurement of collagenase in colonic anastomosis.

Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radiochemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50-70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P less than 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P less than 0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo.

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

Role of collagenase in colonic anastomoses: a reappraisal.

Increased collagenolysis, with reduction in collagen concentration, has been incriminated in the breakdown of colonic anastomoses but previous studies have measured only collagen levels and non-specific collagenolytic activity. Collagenase, the initiating enzyme in collagen degradation, is synthesized on demand and controlled by tissue inhibitor of metalloproteinases (TIMP). Antibodies to collagenase and TIMP were applied to colonic anastomoses in rabbits to investigate the role of the enzyme during healing. Within 12 h of operation, secreting cells and extracellular collagenase were identified at the everted edges of the bowel wall. After 24 h, collagenase activity was accompanied by TIMP secretion in the same localized regions, and by the third postoperative day very few cells were still synthesizing enzyme in these areas, although extracellular activity remained visible. TIMP-secreting cells, however, were seen in a layer of connective tissue sealing the serosal surface of the anastomosis. At 7 days, both enzyme and inhibitor were found only in small aggregates of secreting cells in the deeper layers. The localization and extent of collagenase and TIMP activity accorded well with a normal healing response as, at all times, the enzyme was confined to the immediate vicinity of the suture line.

Tissue culture of retinal glial cells.

In order to be able to investigate the properties and characteristics of glia in the retina, a monotypic culture of retinal glial cells is likely to be an important research vehicle. Several techniques are now available to produce cultures of glial cells from the retina. These methods generally result in cultures of Mueller cells rather than retinal astrocytes. Publications on glial cultures involved complex procedures for the isolation of the target cell. Recent developments have resulted in simpler procedures with the advantage that large numbers of cultures can be established quickly and easily. Glial cultures have already been used in a variety of studies, simpler methods of culture, particularly if these can be adapted for culture of human glial cells, will probably result in more extensive use of cultures to unravel the properties of retinal glia.